1. Field of the Invention
The present invention relates to an apparatus and method employing incoherent light sources, in particular, light emitting diodes, as excitation light sources in a flow cytometer. More particularly, the present invention relates to an apparatus and method employing light emitting diodes as excitation light sources for emitting light toward a particle stream in a flow cytometer to cause particles or cells of interest in the stream to fluoresce so that characteristics of the particles or cells can be examined based on the detected fluorescence.
2. Description of the Related Art
Flow cytometers known in the art are used for analyzing and sorting particles in a fluid sample, such as cells of a blood sample or particles of interest in any other type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer transports the particles (hereinafter called xe2x80x9ccellsxe2x80x9d) in the fluid sample as a cell stream to a flow cell, while also directing the sheath fluid to the flow cell.
Within the flow cell, a liquid sheath is formed around the cell stream to impart a substantially uniform velocity on the cell stream. The flow cell hydrodynamically focuses the cells within the stream to pass through the center of a laser beam. The point at which the cells intersect the laser beam, commonly known as the interrogation point, can be inside or outside the flow cell. As a cell moves through the interrogation point, it causes the laser light to scatter. The laser light also excites components in the cell stream that have fluorescent properties, such as fluorescent markers that have been added to the fluid sample and adhered to certain cells of interest, or fluorescent beads mixed into the stream.
The flow cytometer further includes an appropriate detection system consisting of photomultiplier tubes, photodiodes or other light detecting devices, which are positioned to collect light from the intersection point. The flow cytometer analyzes the detected light to measure physical and fluorescent properties of the cell. The flow cytometer can further sort the cells based on these measured properties.
Known flow cytometers similar to the type described above are described, for example, in U.S. Pat. Nos. 3,960,449, 4,347,935, 4,667,830, 5,464,581, 5,483,469, 5,602,039, 5,643,796 and 5,700,692, the entire contents of each patent being incorporated by reference herein. Other types of known flow cytometer, are the FACSVantage(trademark), FACSort(trademark), FACSCount(trademark), FACScan(trademark) and FACSCalibur(trademark) systems, each manufactured by Becton Dickinson and Company, the assignee of the present invention.
Known flow cytometers, such as those mentioned above and described in the patents cited above, usually employ lasers as the light sources that emit light beams which are directed toward the cell stream to excite particles of interest in the cell stream to cause those particles to fluoresce. Although lasers are generally effective in producing focused beams which are of sufficient intensity to excite the particles of interest to provide detectable fluorescence, the use of lasers can have some drawbacks. For example, the types of lasers employed in many known flow cytometers are very expensive, and thus increase the overall cost of the system.
Also, because the lasers emit very high intensity light, stray light from one of the laser beams can interfere with the fluorescent light emanating from the particles of interest caused by excitation from another laser beam, thus adversely affecting fluorescence measurements. In an attempt to eliminate this problem, a flow cytometer including multiple lasers can be configured to operate the lasers or other light sources, such as arc lamps, in an intermittent or pulsed manner as described, for example, in U.S. Pat. No. 4,573,796 to Martin et al., U.S. Pat. No. 5,185,265 to Steen et al. and U.S. Pat. No. 4,006,360 to Mueller, and in a publication by H. B. Steen and O. J. Sorenson entitled xe2x80x9cPulse Modulation of the Excitation Light Source Boosts the Sensitivity of an Arc Lamp-Based Flow Cytometerxe2x80x9d, Cytometry, Vol. 14, No. 2, pages 115-22 (1993), the entire content of these patents and this publication being incorporated herein by reference. This strobing or pulsing technique is further described in a publication by D. B. Kay and L. L. Wheeless, Jr. entitled xe2x80x9cLaser Stroboscopic Photographyxe2x80x94Technique for Cell Orientation Studies in Flowxe2x80x9d, The Journal of Histochemestry and Cytochemistry, Vol. 24, No. 1, pages 265-268 (1976), in a publication by R. C. Leif and R. A. Thomas entitled xe2x80x9cElectronic Cell-Volume Analysis by Use of the AMAC I Transducerxe2x80x9d, Clinical Chemistry, Vol. 19, No. 8, pages 858-70 (1973), and in a publication by R. A. Thomas, B. F. Cameron and R. C. Lief entitled xe2x80x9cComputer-Based Electronic Cell Volume Analysis with the AMAC II Transducerxe2x80x9d, The Journal of Histochemestry and Cytochemistry, Vol. 22, No. 7, pages 626-41 (1974), the entire contents of each of these publications being incorporated herein by reference.
The techniques described in the documents referenced above have been only partially successful, because the types of lasers and arc lamps having characteristics suitable for use in flow cytometry experience difficulty in being turned on and off rapidly for short periods of time. Therefore, a need exists for an improved system and method which enables a flow cytometer to obtain more accurate measurements while also decreasing the overall size and cost of the instrument.
An object of the present invention is to provide a system and method employing improved excitation light sources for use in a flow cytometer.
Another object of the present invention is to provide a system and method employing light emitting diodes as excitation light sources for use in a flow cytometer to reduce the overall cost of the flow cytometer.
A further object of the present invention is to provide a system and method capable of effectively pulsing the excitation light sources in a flow cytometer to minimize interference between the light emitted by multiple light sources while also decreasing power consumption and increasing the life of the light sources.
These and other object of the present invention are substantially achieved by providing an apparatus and method for examining particles in a flow stream of a flow cytometer, employing incoherent light sources, including incoherent light emitting semiconductor devices such as light emitting diodes (LEDs), and detectors. The light emitting diodes are adapted to operate as the excitation light sources and emit light toward the flow stream, and the detectors detect light, in particular, fluorescent light, emanating from the particles in response to the excitation light striking the particles. The apparatus and method further employs a controller which is adapted to control each of the light emitting diodes to emit their excitation light for a predetermined period during which the excitation light radiates onto particles of interest. The controller evaluates the detected light to ascertain characteristics of the particles, such as particle size, density and granularity. The apparatus and method can further employ one or more coherent light emitting devices, such as a laser, as an additional excitation light source. The detectors can be adapted to detect the LED-excited fluorescence or the laser-excited fluorescence from the particles, and the controller can evaluate both types of detected light to ascertain characteristics of the particles. Furthermore, the controller can control the LEDs to operate in a pulsed manner, which can be synchronized with the detection of the laser-excited fluorescence.